Electron tomography (ET) is an important tool for determining three-dimensional subcellular structures. We have implemented ET in a 300 kV transmission electron microscope to determine the three-dimensional organization of supramolecular assemblies and small organelles in tissues and cells. In general, it is difficult to obtain cryo-electron tomographic data from large tissue samples maintained in their frozen hydrated state. In those cases, useful results can often be obtained by rapidly freezing the cells, freeze-substituting the water for solvent, embedding in plastic and sectioning at room temperature. We have collected dual-axis tilt series from such freeze-substituted specimens and performed 3D reconstructions using the weighted back-projection (WBP) algorithm or the simultaneous iterative reconstruction technique (SIRT). We have applied electron tomography to elucidate the structure of a highly complex supramolecular assembly, the post-synaptic density (PSD), which could eventually lead to a better understanding of neurological diseases. The PSD, which is embedded in the postsynaptic membrane, contains receptors, scaffold molecules, and cytoskeletal elements and is the primary postsynaptic site for signal transduction and signal processing. The PSDs at excitatory synapses contain glutamate receptors of the NMDA and AMPA type. Recycling of AMPA receptors at the PSD accounts for dynamic changes in synaptic transmission. The PSD is known to contain hundreds of different proteins and has been extremely difficult to study by conventional structural techniques. Our electron tomograms recorded from suitably stained freeze-substituted neurons of cultured rat brain showed that PSDs contain vertically oriented filaments, which intertwine with horizontally oriented filaments lying close to the postsynaptic membrane, and define an orthogonal interlinked scaffold at the core of the PSD. The thicket of vertical filaments gives rise to the typical dense appearance that is characteristic of PSDs in standard EM cross-sectional views. The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. We have explored the structural role of MAGUKs at hippocampal excitatory synapses by simultaneous knock down of PSD-95, PSD-93, and synapse-associated protein (SAP)102 and performed TEM tomographic imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-D-aspartate receptors (NMDARs). This knockdown diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane associated PSD-95like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. Our tomographic reconstructions indicate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD. Our results have further demonstrated that ET combined with automated data acquisition in a 300 kV TEM provides useful 3D structural information about the organization of large protein assemblies and organelles in neuronal tissue that is prepared by rapid freezing and freeze-substitution. We have also obtained quantitative 3D information from cells based on conventional transmission electron microscopy by using a stereological approach, and applied this to characterize glycogen distributions in stem cells under different growth conditions.